Copyright 0 1997 by the Genetics Society of America
The Schizosaccharomyces pombe r e d 6 Gene Product
Regulates Multiple Meiotic Events
Ywan Feng L
i'and Gerald R. Smith
Fred Hutchinson Cancm Research Center, Seattle, Washington 981 04
Manuscript received September 26, 1996
Accepted for publication January 15, 1997
ABSTRACT
Previously isolated meiotic recombination (rec) mutants of Schizosaccharomycespombe define 16 complementation groups. The rec genes cloned and sequencedto date reveal little amino acid sequence identity
to other reported proteins. We examined the rec mutants for alterations in meiotic events other than
recombination to gain insightinto therec gene functions and to assess whether they affect recombination
directly or indirectly. While mutations in the rec6-12, 14, 15 and 1 9 genes appeared to affect only
meiotic recombination, a mutation in reel6 delayed meiotic DNA synthesis and, in some instances,
reduced its amount; mitotic DNA synthesis was not detectably altered, indicating that the reel6 effect is
limited to meiosis. In the reel6 mutant some meiotically induced transcripts (e.g., rec7 and 15) were
significantly reduced in abundance, whereas others (e.g., reel0 and exol) were induced and degraded
with normal timing and extent duringmeiosis, indicating that the red 6 mutation leaves the basic meiotic
program intact. These results indicate that the rec genes other than reel6 have their primary effect on
meiotic recombination. In contrast, the reel6 gene product is essential for normal meiotic replication,
recombination, and induction of some transcripts. These meiotic events may be coupled via a dependence of recombination and transcription on replication or via a cascade of gene expression.
S
UCCESSFUL meiosis requires the orderly progression of events, much as the mitotic cell cycle does.
Preceding the two nuclear divisions in meiosis are a
single round of DNA replication, high levels ofrecombination, and induction
of numerous genes. These events
are presumably regulated by genes analogous to those
controlling the mitotic cell cycle. We describe here the
regulation of meiotic events by the reel6 gene product
of Schizosaccharomyces pombe.
To determine the gene functions required for meiotic recombination in S. pombe, our laboratory has isolated mutations (rec) thatreduce or abolish meiotic
recombination. The 39 recessive rec mutations studied
most thoroughlydefine 16 complementationgroups
(PONTICELLI
and SMITH1989; DEVEAUX
et al. 1992).
Based upon their reductions of intragenic recombinant
frequencyatthe
ade6 locus, these rec genes were
grouped into three classes: class I mutations (rec6, 7, 8,
12, 14, and 15) reduce ade6 recombination by a factor
of 1000; class I1 mutations (recl0, 11, and 16), by a
factor of 100; and class 111 mutations (rec9, l?, 17, 18,
19, 20, and 2 1 ) , by a factor of about three to 10. Mutations in swi5 and rad32 also reduce meiotic recombination by a factor of 10 (SCHMIDT
et al. 1987; TAVASSOLI
et al. 1995), and these genes were grouped into class
111. Class I and I1 rec mutants do not show detectable
-
-
-
Corresponding author: Gerald R. Smith, Fred Hutchinson Cancer
Research Center, 1124 Columbia St., Seattle, WA 98104.
E-mail: [email protected]
'Present address: Institute of Biochemistry, National Yang-Ming University, Shih-Pai, Taipei 11221, Taiwan, R.O.C.
Gcnetics 1 4 6 57-67 (May, 1997)
mitotic phenotypes, such as slow growth rate or sensitivity to DNA damaging agents, whereas rec9, 17, and 1 9
mutants (class 111) are sensitive to methyl methanesulfonate and LJV light during mitotic growth. All of these
rec mutations affect meiotic recombination but not, as
far as tested, mitotic recombination.
Cloning and nucleotide sequence analysis of the rec
genes could, by comparison with previously characterized genes and proteins, reveal the functions of the rec
gene products. All of the class I and I1 rec genes, except
recl6, have been cloned and sequenced (LINet al. 1992;
LIN and SMITH1994, 1995a,b; LI et al. 1997; D. H. EVANS, Y . F. LI, M. E. FOXand G. R. SMITH, submitted for
publication). Among these cloned rec genes, only one,
recl2, appears to encode a protein homologous to reported proteins. The deduced Recl2 amino
acid sequence shares limited identity to that of Spoll of Saccharomyces cerevisiae (B. BAUMand Y . LIN,personal communication), and the phenotypes of reel2 and spoll
mutations are similar (KLAPHOLZ et al. 1985; DEVEAUX
et al. 1992). However, the biochemical function of
Spoll is unknown. Therefore, cloning and sequencing
of the rec genes have revealed no further information
on their possible biochemical functions and roles in
recombination.
An alternative approach to reveal the functions of the
rec genes is to characterize the phenotypes of the rec
mutants in as many aspects as possible. The rec mutants
were isolated solely on their deficit in meiotic recombinants (PONTICELLI
and SMITH1989; DEVEAUXet al.
1992). Phenotypes other than recombination and sensi-
Y. F. Li and G. R. Smith
58
TABLE 1
S. pombe strains
Strain
GP13
GP36
GP48
GP59
GP61
GP65
GP273
GP277
GP288
GP289
GP290
GP29 1
GP337
GP449
GP45 1
GP453
GP455
GP458
GP461
GP475
GP477
GP489
GP49 1
GP497
GP499
GP514
GP535
GP571
GP572
GP595
GP611
GP622
GP623
GP624
GP625
GP626
GP627
GP660
GP670
GP674
GP806
GP807
GP813
GP879
GP930
GP1083
GP1646
GP1653
GP1950
GP1951
GP1952
GP1953
GP1956
GP1957
GP1958
GPl967
GP1968
Genotype
h- adeG52
hi adeGM216 patl-114
h' patl-114 endl-458
hi ade6-M26 ura4-294
h- leul-32 endl-458
h+ patl-114
hi ade6-52 rec6-103
hi ade6-52 rec7-102
hi ade6-52 rec9-104
h- adeG52 recl0-109
hi ade6-52 red-110
h- ade6-52 recll-111
h-/hi ade6-M26/ade6-210 argl-2/+ +/ura4-294
leul-32/+ patl-l14/patl-114 endl-458/endl-458
h- ade6-52 rec7-102 pall-114
hi ade6-52 red-1 10 pall-1 14
K ade6-52 recll-111 patl-114
hi ade6-52 rec9-104 pall-114
h- ade6-52 rer10-109patl-114
hi ade6-52 ree9-104 pntl-114 endl-458
Iti ude6-52 rec7-102 patl-114 endl-458
hi ade6-52 rec8-110 pall-114 endl-458
h- a d 6 5 2 rerlO-109patl-ll4 endl-458
h- ndr6-52 rec6-103 patl-114 endl-458
hi ade6-52 recl2-117
h- adeG52 rer14-120
h- adeb-52 rerll-111 patl-114 endl-458
hi ade6-M26 pall-114 endl-458
h- ude6-52 ree15-124
hi add-52 recl6-125
h+ ade6-MZ6 recl6-125
hi ade6-52 recl2-117patl-114 endl-458
h- ade6-52 rec16-125 patl-114 endl-458
h' ade6-52 rec16-125 patl-114 endl-458
h- adr6-52 rec16-125 patl-114 endl-458
hi ade6-52 patl-114 endl-458
h
' ade6-52 patl-114 endl-458
hi ade6-52 rec14-120 patl-114 endl-458
hi ade6-52 recl9-139
h- ade6-52 recl9-139 pall-1 14 endl-458
hi ade6-52 rec15-124 pall-114 endl-458
h- ude6-M26 recl6-125 pall-1 14
h- ade6M26 rec16-125 patl-114 endl-458
h+ ade6-M26 srui5-39 (or 134) patl-114 endl-458
hi add-52 ural-171
h+ adeb"26 lys3-37 prol-l
hi pxol-l::ura4+ pall-1 14 endl-458 leul-32 ura4D18
h- adeGM26 urnl-171 rec16-125
h+ ade6-52 lys3-37 prol-1 red 6-125
adeG52 recl6-125 patl-I14 endl-458
h- ade6-52 rrcl6-125 pall-114 a d l - 4 5 8
h+ ade6-52 rec16-125 lys3-37fn-01-l patl-114 endl-458
h+ ade6-52 lys3-37 prol-l patl-114 endl-458
h- ade6-MZ6 reel6125 ural-171 patl-114 endl-458
hi adeGM26 ural-171 pall-114 endl-458
h- ade6-MZ6 ural-171 patl-114 rec16-125
hi ade6-M26 ura4-294 patl-114 endl-458 rec16-125
hi ade6-M26 ura4-294 patl-114 endl-458
h +
Source"
PONTICELLI
and SMITH(1989)
A. KLAR, strain SP301, (IINOand YAMAMOTO1985a)
A. PONTICELLI"
A. P O N T I C E I ~
SZANKASI
and SMITH(1992)
A. PONTI(:EI.I,I~
and SMITH(1989)
PONTICELLI
PONTICEILI
and SMITH(1989)
PONTICELLI
and SMITH (1989)
and SMITH(1989)
PONTIC:ELLI
PONTICELLI
and SMITH(1989)
and SMITH (1989)
PONTICELLI
A. PONTICELLI~
GP65 X GP277
GP65 X GP290
GP65 X GP291
GP65 X GP288
GP65 X GP289
GP455 X GP48
GP449 X GP48
GP451 X GP48
GP458 X GP48
GP273 X GP48
DEVEAUX
et al. (1992)
et al. (1992)
DEVEAUX
GP453 X GP48
SZANKASI
and SMITH(1992)
DEVEAUXet al. (1992)
DEVEAUX
et al. (1992)
et al. (1992)
DEVEAI~X
GP497 X GP48
GP572 X GP48
GP572 X GP48
GP572 X GP48
LINand SMITH (1994)
GP48 X GP13
GP499 X GP48
DEVEALTX
et al. (1992)
GP660 X GP48
GP571 X GP48
GP595 X GP622
GP595 X GP622
J. VIRGIN''(SGHMIDT
et al. 1987)
DEVEAUX
and SMITH(1994)
and SMITH(1994)
DEVEAUX
and SMITH1995)
P. SZANKASI~
(SZANKASI
GP879 X GP595
GP930 X GP572
GP623 X GP535
GP623 X GP535
GP1653 X PG535
GP1653 X GP535
GP1646 X GP626
GP1646 X GP626
GP1646 X GP626
GP623 X GP59
GP623 X GP59
59
Regulation of S. pombe Meiotic Events
TABLE 1
Continued
Strain
GP1977
GP1979
Genotype
hi/ h- ade6-52/adef3"26 recl6-125/wcl6-125 lys3-37/ +
p r o l - l / + +/ural-l71 patl-l14/patl-114 endl-458/
endl-458
h-/h- ade6-52/ade6"26 lys3-37/+ p r o l - I / +
+/ural-l71 patl-114/patl-I14 endl-458/mdI-458
GP1952
X
GP1956
GP1953 X GP1957
X, segregant from a meiotic cross or diploid from conjugation of the indicated parents. Y. LIN constructed GP strains 611,
622, 623, 624, 625, 626, 627, 670, 674; P. SZANliASI , GP449,451,453,455,458, 461, 475,477,489,491,514, 1083; and J. VIRGIN,
GP806, 807, 813.
' Genealogy available upon request.
tivity to DNA damaging agents have not been reported,
except for aberrancies in chromosome segregation and
axial elements of meiotically paired chromosomes in a
recarnutant (MOLNARet al. 1995). Therecgene functions
might be directly involved in recombination, in which
case other aspects of meiosis might be unaltered in the
mutants. Alternatively, the rec gene functions might affect other aspects of meiosis, such as DNA synthesis or
regulation of gene expression, and only indirectly affect
recombination. To test these alternatives, we studied the
synthesis of DNA, the induction, splicing, and degradation of transcripts, and thetiming of the meiotic divisions
and spore formation in the rec mutants.
To analyze events during meiosis, it is important to
have synchronized meiotic cells. Synchrony is most
readily achieved by using the temperature-sensitive allele patl-114 (IINO andYAMAMOTO1985a). The patl'
(= rani+) gene encodes a protein kinase homologue
(MCLEOD
and BEACH1986) that represses meiosis. Inactivation of the patl-114 gene product at high temperature bypasses two essential requirements for S. pornbe
meiosis: heterozygosity at the mating-type locus and nitrogen starvation (IINO and YAMAMOTO 1985a; N U R ~ E
1985). Therefore, patl-214 mutants undergo many aspects of meiosis at high temperature, even in the haploid state.Haploid patl-114 thermally induced cells
form abundant spores, but only -3% of these are viable, due to the deficiency of chromosomes in a haploid
(IINO and YAMAMOTO1985a). Thermally induced patlI14 diploids produceabundant
spores with high
(>50%) viability. Thermally induced meiosis in the
patl-114mutant is well synchronized (BEACHel al. 1985;
IINo and YAMAMOTO1985b; BAHLERet al. 1991; SZANKASI and SMITH 1992), and
the kinetics of meiotic DNA
synthesis in a pat1 haploid strain induced by temperature shift mimics that of a p a t l f diploid strain induced
by nutritional depletion (BEACHet al. 1985). In patl114 haploid cells transcripts of the rec6, 7, 8 ,IO, 11,
12, and 15, and ex01 genes are sharply induced, and
exonuclease I activity sharply rises and falls during meiosis (LIN et al. 1992; SZANKASI
and SMITH1992; LIN
and SMITH1994, 1995a,b; LI et al. 1997). The ade6-M26
meiotic recombinationhotspot
(GUTZ 1971; PONTI-
CELI,I et al. 1988) stimulates recombination both in starvation-induced pall+ diploid meiosis and in thermally
induced patl-114 haploid meiosis (H. CLARKE,Y. F. LI,
J. B. VIRGIN
and G. R. SMITH, unpublished
data). Therefore, meiotic events appear to be similar in pat1 haploid
and patl+ diploid cells. Our initial characterizations of
meiosis in the rec mutants thus used pat1 haploid cells.
The results reported here indicate that the reclbgene
product regulates multiple events in meiosis, whereas
the other rec gene products appear to be directly involved in recombination.
MATERIALS AND METHODS
S.p m b e strains: The strains and their genotypes are listed
in Table 1.
Meiotic crosses and recombinantfrequency detennination: The procedures for meiotic crosses and determination
of meiotic recombinant frequencieswere as described (PONTICELLI and SMITH 1989).
S. pombe media: YEA,YEL and SPA are yeast extract agar,
yeast extract liquid medium, and sporulation agar, respectively (GLITZet nl. 1974). NBA is an agar minimal medium
(PONTICELLI
and SMITH1989) and EMM2* medium is a liquid
minimal medium (SZANKASI
and SMITH1992). PM is similar
to EMM2* minimal medium (BEACHet nl. 1985). PM-N is the
same as PM medium but without NH,CI. Minimal media were
supplemented with required nutrients at 75 yg/ml, except as
noted.
Preparation of meiotic cells: Three methods were used.
(1) Small (-25 ml) cultures of p a t l - I 1 4 haploid and diploid
strains, for measuring DNA synthesis andcommitment to
spore formation andfor
microscopic examination, were
grown at 25" to -5 X IO6 cells/ml in PM medium with adenine
(75 pg/ml), starved for nitrogen for 16 hr in PM-N medium
with adenine (10 pg/ml) to bring the cells to the G1 phase
of the cell cycle, and induced for meiosis by raising the temperature to 34" and restoring nitrogen (BEACHet al. 1985).
To measure the time of commitment to sporeformation (Figure 5), 2-ml samples were removed at the indicated times (04 hr) and incubated at 25" until 12 hr after the initiation of
induction. Material from 1 mlof culture was collected by
centrifugation, suspended in 0.6% glusulase (DuPont), and
incubated overnight at 25". Appropriate dilutions were plated
on YEA and incubated for 6 days at 25" to determine the
concentration of glusulase-resistant colony-forming units
(spores). (2) Small (-25 ml) cultures of pall+ diploid strains
were subcultured for 3 days in YEL medium and grown in
YEL medium at 32" to -1 X IO7 cells per ml; the cells were
60
Y. F. Li and G. R. Smith
TABLE 2
A recl6 mutation reduces recombination independent of temperature,
patl function, or ploidy
Ade+ recombinants/ lo6 viable spores
Strains cxamined
Temperature of mating and meiosis
mated"
A. Haploids
35"
30"
Experiment
25"
20"
1800
4600
2400
58
90
1
GP13 (Tee+) X GP595 ( r e c l 6 )
GP13 (Tee+) X GP595 ( r e c l b )
GP572 (reclh) X GP595 ( r e c l 6 )
GP572 ( r e c l 6 ) X GP595 ( r e c l 6 )
2
1
2
2500
3300
210
70
B. Diploid sporulatedb
Temperature
Experiment
GP1979
GP1977
GP1943
GP1942
GP1943
GP1942
3800
120
120
1
34"
34"
34"
34"
30"
30"
( h - / h - p a t l / p a t l rec+/rec+)
( h - / h - p art le/cplabt/lr e c l 6 )
( h - / h + rec+/rec+)
(h-/h+ reclb/recl6)
( h - / h + rec+/rec+)
( h - / h + reclb/recl6)
C . patl haploid induced"
GP1968 (pade6-469) (rec')
GP1967 (pade6-469) ( r e c l 6 )
2200
80
660
62
1500
72
1400
1100
24
<70
Experiment 2
2500
130
1700
310
6000
170
Experiment 1
Experiment 2
66,000
130
23,000
90
"Strains, which contain the ade6-M26 or ade6-52 mutation, were mated on supplemented SPA medium at
the indicated temperature. Spores were harvested and plated on YEA for total spores and on NBA without
adenine (experiment 1) or YEA f guanine (experiment 2) for Ade' recombinants.
These diploids contain the ade6-M26 and ade6-52 mutations. The p a t l strains were induced for meiosis by
raising the temperature to 34" (method 1; MATERIALS AND METHODS). After 24 hr spores were harvested and
assayed for Ade' recombinants. The cultures in experiment 1 were used for the experiment in Figure 3. The
p a t l + strains were induced for meiosis by starvation for nitrogen (method 2; MATERIALS AND METHODS) at 34"
or 30", as indicated, for 24 hr and assayed for Adef recombinant spores.
'These strains contain the ade6-M26 mutation on the chromosome and the
ade6-469 mutation on the
plasmid. They were induced for meiosis by raising the temperature to 34" (method 1 ) and after 24 hr assayed
for Ade+ recombinant spores.
collected by centrifugation and resuspendedin PM-N medium to inducemeiosis (BAHLER
et al. 1993). (3) Large (-500
ml) cultures of patl-114 haploid strains, for measuring RNA
synthesis, were grown at 25" in YEL medium to saturation,
diluted into EMMP* to 0.D.600 = 0.05, grown to 0.D.600=
0.3, and induced for meiosis by raising the temperature to
34" (SZANKASI
and SMITH1992). Cultures of ret+ and 'rec mutant cells were always analyzed side-by-side.
DNA content measurement: The DNA content of cells was
determined by flow cytometry according to an unpublished
laboratory manual of PAUL NURSE. Approximately 1 X 10' cells
were collected by centrifugation and resuspendedin 1 ml
of cold 70% ethanol. For each measurement, 0.3 ml of this
suspension was washed in 0.5 ml of 50 mM sodium citrate
(pH 7.0) and resuspended in 0.5 ml of this buffer containing
RNase A (0.1 mg/ml). The samples were incubated at 37" for
2 hr. One-half milliliter of propidiumiodide(4pg/ml;
Sigma) in sodium citratewas added to the samples. After brief
sonication to disruptcell clumps, the fluorescence of 10,000
cells was determined by flow cytometry using a Becton Dickinson FACScan.
Microscopic analysis: About 100-200 cells fixed with ethanol and stained with propidium iodide as described above
were examined with a Nikon Microphot EPI-FL microscope
with a PlanApo 60 objective lens.
Northern blot hybridization: Unfractionated RNA was prepared from thermally induced meiotic cultures of pat1 haploid strains (S7ANKASI and SMITH1992) and analyzed by
Northern blot hybridization as described (LIN et al. 1992).
DNA fragments containing a rec gene ora plasmid containing
-
the ex01 gene were used as radioactive probes to detect transcripts as follows: rec6, 0.45-kb SacI-PstI fragment ofpYL63
(LIN andSMITH1994); rec7, 0.37-kb Pstl-Sty1 fragment of pYLl
(LIN et al. 1992); ree8, 0.9-kb BstNI fragment of pYL3 (LINet
al. 1992); rec10, 2.2-kb Sac1 fragment of pYL177 (LIN and
SMITH199513); recll, 2.5-kb EcoRI-Sa& fragment of pYFL102
(LI et al. 1997); recl2, 0.74kb NheI-PuuII fragment of pYL122
(LIN and SMITH1994); recl5, 0.61-kb EcoRV-PuuII fragment
of pYL157 (LIN andSMITH1995a); exol, pXON401, a derivative of plasmid pBluescript I1 SK( +) containing theex01 gene
( SZANKASI
and SMITH 1995).
Hybridization signals were quantitated using a PhosphorImager model 400E (Molecular Dynamics).
RESULTS
Recombination deficiency of the r e d 6 mutant is not
affected by the temperature,patl function or ploidy of
the cells: Our analysis of meiotic events in rec mutant
cells typically used putl-I14 (Ts) haploid cells induced
for meiosis by raising the temperature to 34". Our previous analyses of recombination, however, used putl' d i p
loid cells induced for meiosis by starvation for nitrogen
at 25" or 30". Because red6 mutants had alterations in
DNA and RNA metabolism under the former conditions (patl-124 cells at 34"; see below), we tested
whether the alteration of recombination by the rerl6-
61
Regulation of S. pombe Meiotic Events
Time (h)
GP622 (rec76)
after meiotic
induction
i
0
80
1
60
A
L
L
1
40
-a-
20
-0-
*
GP625 @-,haploid)
GP626 (hi, haploid)
GP337 (h-h-,diploid)
A
2
-
3
4
5
U
"
0
4
2
Time (hour) after meiotic induction
IUU I
6
/
20
ba
-A-H-0-
I
1
U
4
GP625 (re&)
GP475 (recl)
GP489 (reclo)
GP611 (recla
GP674 (recl5j
I
T
0
2
Time (hour) after meiotic induction
FIGURE
1.-Meiotic DNA synthesis in haploid and diploid
patl cells. (A) ret' strains. (B) Haploid ret+ and rec mutant
strains. Cells were grown and induced for meiosis by raising
the temperature to 34" (Method 1; MATERIALSAND METHODS).
Sampleswerewithdrawn at the times indicated, fixedwith
ethanol, treated with RNase, and stained with propidium iodide. The percentage of cells with DNA content of 2c (for
the haploid strains) or 4c (for the diploid strain) was determined byflow cytometry (see Figure 2A for an example).
Samples at 0 hr were taken immediately before the temperature was raised to 34".
125mutation, the only recl6 allele isolated to date
(DEV E ~ U et
X al. 1992), depended on the temperature,pat1
allele, o r ploidy of the cells. Standard matings of heterothallic ade6-M26 and ade6-52 strains were conducted at
20-35", and the spores were tested for Ade+ recombinant frequency. The
recl6 mutation reduced the recombinant frequency by a factor of 20-35 regardless of
the temperature (Table 2A). Recombinant frequencies
were slightly reduced (by a factor of two or three) at
the highest and lowest temperatures in both the ret+
a n d rec16 crosses.
I
l
l
2c
IC
2c
DNA content
B
-Q-
I
IC
loo
,
I
i
0
"e
2
4
GP622 (rec7k)
6
8
Time (h) after meiotic induction
FIGURE
2.-Meiotic DNA synthesis in haploidpatl rec" and
r e d 6 mutant cells. (A) Flow cytometric profiles of patl ret'
and p a t l r e d 6 mutant cells. The experiment was performed
as described in Figure 1 and MATERIALS AND METHODS. On
the abscissa ICand 2c represent the DNA content before and
after DNA replication, respectively. The fraction of cells in
each interval of DNA content is represented on the ordinate.
Approximately 10,000 cellswereanalyzed for each sample
taken at the indicated time after the cultures were shifted to
34". (B) Quantitation of the results from A.
To test the effect of the patl allele, we constructed
diploids and sporulated them by raising the temperature to 34" (patl-114 diploids) o r by starving them for
nitrogen at 34" o r 30" (patlf diploids). In each experimental comparison (rec' us. recl6) the r e d 6 mutation
reduced the recombinant frequency, by a factor ranging from six to 35, regardless of the temperature, pat1
allele, or method of meiotic induction (Table 2B).
To test recombination in pat1 mutant haploids, the
cell types used for most of the experiments reported
below, we transformed ade6M26 strains with a plasmid
bearing the ade6-469mutation, sporulated them at 34",
Y. F. Li and G. R. Smith
62
6o
-I
I
20
GP1979 (re&)
GP1977 (rec76)
0 1
0
I
I
I
I
2
4
6
8
Time (h) after meiotic induction
10
FIGURE 3.-Meiotic DNA synthesis in diploid pat1 rec" and
r e d 6 mutant cells. The experiment was performed as described in Figure 1 and MATERIAIS AND METHODS.
and measured Ade+ recombinants among the viable
spores produced by the haploids. The reel6 mutation
reduced the recombinantfrequency by a factor of 250500 (Table 2C). Ade+ recombinant frequenciesin the
reef strain are -10-fold higher than in standard matings, presumably due to the high copy number of the
plasmid (PONTICELLI and
SMITH1989).
In summary, the rec16-125 mutation strongly reduced
recombination regardless of the temperature, patl allele or ploidy of the cells. This outcome permits comparison of ret+ and reel6 mutants under a varietyof
conditions.
Meiotic DNA synthesis is delayed but
not abolished in
a r e d 6 mutant: To examine whether the w e mutations
affect meiotic events other than recombination, DNA
synthesis, an early event in meiosis, was first examined.
Synchronized cultures, requiredto measure the kinetics
of DNA synthesis, were obtained by arresting cells at
the G1 stage through nitrogen starvation (NURSEand
THURIAUX
1977). Meiosis was then induced in the patl114 strains by raising the temperature from 24" to 34"
and restoring a nitrogen source.The DNA content per
cell was measured byflow cytometry at various times
after inductionof meiosis. The reef diploid strain GP337
andthe ret+ haploid strains GP625 ( h - ) and GP626
( h+) underwent one roundof DNAreplication after the
temperature shift. The bulk of DNA synthesis occurred
between 1 and 3 hr after the temperature shift, and by
4 hr -90% of the cells had replicated their DNA once
(Figure 1A). Thekinetics of DNA synthesis in the three
strains was very similar. Therefore, DNA synthesis was
not significantly affected by the ploidy or the mating
type of the cells.
Ten of the rec mutants, as patl haploids, were analyzed in this way for meiotic DNA synthesis. They included all of the class I (recb, 7, 8, 12, 14, and 15) and
classI1 (recl0,11, and 16) mutants, and the class I11
mutant rec9. (The strains analyzed were those listed in
Table 3 and Figure 1B.) The results for the rec7, IO, 12,
and 15 mutants are shown in Figure 1B. The kinetics
of DNA synthesis in all 10 mutants, except recl6, was
not significantly different from thatin reef cells. In contrast, the initiation ofDNA synthesis in recl6 mutant
cells was delayed by about 2 hr relative to that in ret+
cells (Figure 2).
To test whether this delay was caused by the recl6-I25
mutation or by another unidentified mutation,progeny
from a test cross between strains GP623 (reel6 patl)
and GP535 (ret' p a t l ) were analyzed. DNA synthesis
was delayed in all 16 reel6 segregants tested, including
strains GP1950 and 1951 (identified by their deficiency
in nde6 intragenic recombination), and normal in all
16 rut" segregants tested (data not shown). Six additional recl6 patl strains from other crosses were also
analyzed (strains GP622,624,806,807,1956, and 1958);
none showed separation of the phenotypesof DNA synthesis delay and recombination deficiency (datanot
shown). Therefore, thedelay in meiotic DNA synthesis
is caused by the reclb-125 mutation or another closely
linked mutation.
The preceding analysis used haploid strains. To determine whether thedelay of DNA synthesis by the recl6
mutation occurred in diploid
strains, we constructed
diploid patl reef and reel6 strains homozygous for mating type ( h - / h - ) and analyzed them for meiotic DNA
synthesis. The bulk of the DNA synthesis occurred between 2 and 3 hr in the reef cells, but between 3 and 7
hr in the reel6 mutant cells (Figure 3). Furthermore,
only about half of the reel6 mutant cells completed
meiotic DNA synthesis. This result is consistent with the
observation that in these diploid patl recl6cultures only
-50% of the cells completed both meiotic divisions
(data not shown). DNA synthesis was similarly delayed
and reduced in three other reel6 diploid strains (h+/
h+ or h - / h - ) (data not shown).DNA synthesis was thus
delayed and, in diploids, reduced by the recl6mutation.
We attempted to determine the effect of the reel6
mutation on DNA synthesis in patl+ diploids ( h + / h - ) ,
using two methods to induce meiosis by starvation
(BEACHet nl. 1985; BAHLERet al. 1991). Although DNA
synthesis appeared to be somewhat delayed by the reel6
mutation, the low degree of synchrony in these starved
cultures precluded a firm conclusion (data not shown).
Mitotic DNA synthesisis normal in the r e d 6 mutant: Since the reel6 mutation delayed meiotic DNA
synthesis, it might affect DNA synthesis in general.
Therefore, mitotic DNA synthesis was measured in both
patl and patl+ haploid strains. The procedure for synchronization of cells and analysis of DNA synthesis during mitosis was similar to that during meiosis. Mitotic
DNA synthesis in the reel6 mutant cells was essentially
the same as that in ret+ cells both in patl+ strains at
25", 30", and 34" and in patl mutant strains at 25", a
temperature permissive for mitotic growth (datanot
Regulation of S. pombe Meiotic Events
-A-
63
GP475 (recl)
_cf_
GP625 ( r e d )
4
GP625 ( r e d )
+GP1951 (rec76)
tn
S
+GP1951 (rec76)
10'
0
2
4
6
8
10
0
2
4
6
8
10
12
Time (h) at 34oC
Time (hour) after meiotic induction
FIGURE4.-Completion of meioticdivisions in ret+, rec7,
10, 12, 15, and 16mutant cells. Samples of thermally induced
p a t l haploid cultureswere prepared and stainedwith propidium iodide as in Figure 1, and "100-200 cells were examined
with a fluorescencemicroscope. For details see MATERIALS
AND METHODS.
FIGURE5.-Commitment to spore formation in reef and
r e d 6 mutants. Cells were grown and induced for meiosis by
raising the temperature to 34" (Method 1; MATERIALS AND
METHODS). At the indicated times samples were removed and
incubated at room temperatureuntil 12 hr after the induction
of meiosis. The cellswere treated with glusulase to kill vegetative cells and titered on YEA at 25". For details see MATERIALS
AND METHODS.
shown). Therefore, thereel6 mutation affected meiotic
but not mitotic DNA synthesis.
Both meiotic divisions are completed in the rec mutants: In S. pombe meiosis, DNA synthesis is followed by
two sequential nuclear divisions. Cells that have completed both divisions contain three or four nuclei in
pat1 haploid strains (IINO andYAMAMOTO1985a). Nuclei in induced patl rec cells were visualized by microscopy after staining their DNA with the fluorescent dye
propidium iodide. The fraction of cells with three or
four nuclei started to increase between 4 and 5 hr and
reached -80% between -6 and 8 hr after meiotic induction (Figure4). Cells containing threeor fourdense
nuclei (nascent spores within an ascus) became visible
at this time, too. All of the rec strains examined (reef,
rec6, 7, 8, 9, 10, 11, 12, 15, and 16) had a similar timing
and final extent of cells withthree or four
nuclei. Representative results for the rec+, 7, 10, 12, 15 and 16 mutantsare
shown in Figure 4. Additional data (not
shown) suggest that thedifferences in the time of spore
formation among the strains is not significant. Therefore, the rec mutants examined here, including recZ6,
completed the second meiotic division ataboutthe
same time as did rec" cells.
Commitment to spore formation is not delayed
in the
red6 mutant: The occurrence of the second nuclear
division at about the same time in the reel6 mutant as
in the ret' cells indicated that the late steps of meiosis
proceed on schedule in the reel6 mutant. To assess if
earlier steps of meiosis proceed on schedule, we determined the time of commitment to spore formation in
patl-114 reef and reel6 mutants. For this experiment,
commitment to spore formationis defined as the time
at which the thermally induced patl-114 mutant cells
can be returnedto low temperature andstill form glusulase-resistant colony-forming units (spores)after further incubation at low temperature. Without the temperature being raised, there was a verylowlevelof
spores in the cultures,
of that achieved after the
temperature was raised (Figure 5). In both the rec' and
reel6 haploid patl cultures a rapid, extensive rise in the
number of committed cells occurred between 2.5 and
4 hr after induction. No significant difference between
the ret+ and reel6 cultures was seen. The viability of the
spores from recl6 mutant cells was about the same as
that from recf cells -2-3%, similar to that reported by
IINO and YAMAMOTO (1985a).These results indicate
that an event early in meiosis, at -3 hr after induction
(or -1 hr after the initiation of meiotic DNA synthesis
in ret' cells), occurs on schedule in the reel6 mutant.
[In patl+ cells the commitment to meiosis also occurs
at about thetime of DNAsynthesis (BEACHet al. 1985).]
Induction of some rec gene transcripts is reduced in
the recl6mutant: In reef cells transcripts of the rec6, 7,
8, 10, 11, 12, and 15 genes accumulate between 2 and
3 hr after meiotic induction and then disappear (LIN
et al. 1992; LINand SMITH1994, 1995a,b; LI et al. 1997;
Figure 6). To test whether a rec gene product regulates
the expression of one or more rec gene(s), and thus
might indirectly affect recombination, transcripts of the
rec genes cloned to date were examined in several rec
mutants during meiosis. RNA was prepared from patl
thermally induced meiotic cultures of rec strains at 0 , 2,
3, and 4 hr. Transcripts of the rec6, 7, 8, 10, 11, 12, 15,
and ex01 genes were examined by Northern blot hybrid-
64
Y. F. Li and G. R. Smith
FIGURE6.-Transcription of rec and ex01 genes in ref+,
recl4, and reclbmutantcells. (A) Northern blot hybridization.
Unfractionated RNA was prepared from pat1 haploid strains
GP625 (rer+),GP627 (recl4),and GP1951 (reclfj)at the times
indicated after thermal induction of meiosis. RNA (12 pg)
was electrophoresed on a 6% formaldehyde-1% agarose gel
and transferred to a supported nitrocellulose filter. The filter
was probed with fragments of the recor ex01 genes as indicated
~ ~ transcripts
1 5
have hZr0
(See MATERIAL.ANDMETHODS).
forms (LIN and SMITH1995a). (B) Quantitation of wr7, IO,
and 15 and ex01 transcripts by Phosphorlmager analysis. The
maximal intensity for each transcript is set at 1, and others
are expressed relative to it. The slower migrating rec15 transcripts are shown here.
ization. The S. pornbe Ex01 protein acts in mismatch repair, and transcripts of the ex01 gene are meiotically
induced between 3 and 6 hr ( S ~ ~ K A
and
S ISMITH1995),
an induction pattern different from that of the reported
rec genes. Therefore, the ex01 gene serves as a control in
this study. The q c gene transcripts, whose abundance
changes less than twofold during meiosis (LIN et al.
1992), were also examined in all cases as a control for
RNA abundance and integTitV (data not shown).
Transcripts of all of the rec genes examined accumulated between 2 and 3 hr in all of the w c mutants, except
rec16, with the same kinetics and to the same extent as
in ret+ cells and disappeared at 4 hr (Table 3). Representative results are shown in Figure 6.
In the we16 mutant, some transcripts accumulated
and disappeared as in ret+ cells, while other transcripts
were only weakly induced (Figure 6). For the reclOand
ex01 transcripts the timing and extent of accumulation
were the same in the reel6 mutant as in the rpc' cells
or the reel4 mutant, a representative mutant shown in
Figure 6. These results indicate that the progression of
meiosis was not delayed in the reel6 mutant, as indicated previously by the timing of the meiotic divisions
(Figure 4) and the commitment to spore formation
(Figure 5 ) . In contrast, transcripts of rec7 and 15 were
reduced in abundance by a factor of about seven to 10
in the reel6 mutant compared to we+ cells or the reel4
mutant (Figure 6B). Transcripts of rpc6, 8,and I 1 were
reduced in abundance by a factor oftwo to three in
the r ~ 1 mutant,
6
but thetiming of their accumulation
and disappearance was similar to that in the rpr' cells
or other rp(: mutants (Figure 6 and data not shown).
Due to the nature of the Northern hybridization analysis, these two- to threefold differences may not be significant. Similar results were obtained with a different
reel6 haploid (strain GP622) and with a reel6 diploid
(strain GP1977) (data not shown).
In summary, the accumulation of meiotic transcripts
occurred with normal kinetics and to the normal level
in all of the recmutants examined exceptrecl6, in which
some transcripts were reduced in amount while others
were normal.
The rec mutations do not affect splicingof the recZ5
transcripts: The rpcI5 transcript has a 49-bp intron,
which is removed during meiosis (LINand SMITH
1995a). To test whether splicing of the reel5 primary
transcripts is regulated by other rec gene products, RNA
was prepared from p n f l rec mutants at 0 and 2 hr after
meiotic induction and examined by a reverse transcrip
tion-polymerase chain reaction (RT-PCR) and gel electrophoresis analysis for reel5 transcript splicing (LIN
and SMITH1995a). Splicing of reel5 transcripts occurred
in each of the rec mutants tested (listed in Table 3) as
well as in srui5 and ex01 mutants and in ret+ cells (data
not shown). Therefore,splicing of the rec15 transcripts
did not depend on the rpc gene products examined
here.
DISCUSSION
Most rec mutations affect meiotic recombination but
notothermeioticeventsexamined
Previous studies
showed that the rpc mutations strongly reduce meiotic
recombination, by factors of up to 1000 (PONTICELLI
and SMITH1989; DEVFAUX
et nl. 1992). The results reDorted here show that, except for rpcl6, the rec muta-
65
Regulation of S . pornbe Meiotic Events
TABLE 3
Accumulation and degradation of rec and exol transcripts in rec mutants
Transcripts"
Strain
GP625 (ret+)
GP49 1 ( rec6)
GP475 (rec7)
GP477 ( r e d )
GP461 (re&)
GP489 ( r e f l o )
GP514 ( w e l l )
GP611 ( r e c l 2 )
GP627 ( r p c l 4 )
GP674 ( r e c l 5 )
GP1951 ( r e c l 6 )
GP670 ( r e c l 9 )
GP813 (s7ui5)
GP1083 ( e x o l )
rec6
rec 7
rec8
reel 0
reel I
reel 2
red5
ex01
+
+
+
+
+
+
+
+
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
i-
+
+
+
-
ND
+
+
+
+
+
+
+
+
?
+
+
+
+
+
+
+
+
+
+
+
+
ND
ND
+
+
+
+
ND
+
+
+
+
+
+
t
ND
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
"Transcripts of the recand ex01 genes in therec, swi5, and exol strains were analyzed by Northern blot hybridization. Unfractionated RNAwas prepared at 0, 2, 3, and 4 hr from thermally induced meiotic cultures (method 3; MATERIALS AND METHODS) of
the pat1 haploid strains indicated in the left column. The radioactive probes used to detect transcripts are indicated in the top
row (see MATERIALS AND METHODS for details). The results were obtained from one to three independent experiments. See
Figure 6 for representative results. +, the accumulation and degradation of transcripts was similar to that in the rec" strain; ?,
the accumulation of transcripts was reduced by a factor of two to three; -, the accumulation of transcripts was significantly
reduced; ND, not determined.
tions tested have no significant effect on the othermeiotic events examined. These events include the timing
and extentof meiotic DNA synthesis (Figure 1 and data
not shown), the accumulation and degradation of rec
and exol transcripts (Figure 6 and Table 3), the splicing
of meiotically induced reel5 transcripts (datanot
shown), and the secondmeiotic division (Figure 4 and
data not shown). Since recombination, but not other
events tested, is affected, these observations indicate
that these rec gene products (rec6-12, 14, 15, and 19)
act more directly in recombination than in other meiotic events. These rec gene products may act only in
recombination.
The rec genes in this set do not, however, have the
same roles in recombination. The three class I11 mutants examined (rec9, rec19, and swi5) have only modestly reduced meiotic recombination (factors of 10 or
less), whereas the class I (rec6, 7, 8, 12, 14, and 15) and
class 11 (reel0 and 11) mutants have more strongly
reducedrecombination(factors
of 100-1 000)
(SCHMIDT
et al. 1987; PONTICELLI and
SMITH1989; DEVEAUXet al. 1992). The rec8, 10, and 11 gene products
activate recombinationina
region-specific fashion,
more strongly on chromosome IIIthan onchromosome
I or ZI (DEVEAUX
and SMITH 1994). The
rec6, 7, 12, 14,
and 15 gene productsare required for meiotic recombination in all intervals tested and may be required
throughout the genome
(DEVEAUXand SMITH1994;
LINand SMITH1994, 1995a; R. DING,D. H. EVANS,Y. F.
LI, Y. LINand G. R. SMITH, unpublisheddata). Further
phenotypic analysis of these rec mutants may reveal dif-
ferences among them and provide clues to their functions.
The red6 mutation affects meiotic recombination,
replication, and some rec gene transcription: The rec16125 mutation, the only reel6 mutation isolated to date,
was unique in our survey. Meiotic DNA replication was
delayed by -2 hr and, in diploids, reduced in amount
in pat1 thermally induced meiosis (Figure 2 and 3). In
p a l l f starvation-induced meiosis of diploids the delay
was less clear (data not shown), but the
delay may have
been obscured by the lower degree of synchrony in
patl' starvation-induced meiosis than in pat1 thermally
induced meiosis.
Transcripts of the rec7 and 15 genes accumulated to
high levels between 2 and 3 hr after induction of meiosis and then disappeared in reef cells (LIN et al. 1992;
LIN andSMITH1995a; Figure 6), but they accumulated
to only low levels, barely above the uninduced level, in
the reel6 mutant (Figure 6). These observations suggest
that transcription of re67 and I 5 is induced in meiosis
and thatthis induction requires therecl6' gene product
(Recl6). Not all meiotically induced genes, however,
require Recl6: transcripts of rer10 and ex01 accumulated at the same time and to about the same extent in
the reel6 mutant as in ret+ cells (Figure 6 ) . A slight, but
perhaps not significant, reduction was observed for red,
8 and 11 transcripts. Thus, Recl6 appears to be required for meiotic induction of some, but not other,
genes. We have not determined whether these inductions involve increased transcript synthesis or stabilization, or both.
66
Y . F. Li and G. R. Smith
The alterations in thetiming of meiotic DNA synthesis and in the extent of accumulation of transcripts in
the recl6 mutant do not appear to
result from a disruption of the meiotic program. Several meiotic events occurred essentially on schedule. These events include
induction of the reel0 and ex01 transcripts (Figure 6),
degradation of reel0 transcripts (Figure 6), commitment to spore formation (Figure 5), and the second
meiotic division (Figure 4). Thus, the delay in meiotic
DNA synthesis, the reduced levels of rec7 and 15 transcript abundance, and reduced recombination do not
stem from failure of reel6 mutant cells to progress
through meiosis at the normal rate.
We propose two, not mutually exclusive, explanations
for the role of Recl6 in coupling meiotic recombination, replication and transcription. The first explanation supposes that Recl6 directly controlsthe expression of certain meiotically induced genes, or the
activities of their products,which in turn control
recombination and replication. In this view there are two sets
of meiotically induced genes. Some, such as rec7 and
15, require Recl6, whereas others, such as recl0 and
exol, do not. The products of some of the Rec16-controlled genes (e.g., rec7 and 15) are demonstrably required for meiotic recombination, whereas others may
be required forreplication. Still others may be required
for transcription of additional genes. This type of meiotic control is manifest by the pat1 and mei2 genes,
whose products control induction of the meiotic program, including meiotic replication and transcription
(reviewed by EGELet al. 1990; WATANABE
and YAMMOTO 1994). For example, when Pat1 is inactivated, the
rep1 gene is induced, and its product is required for
meiotic DNA synthesis and fortranscription of the meiotically induced res2 and cdc22 genes (SUGNAMA et al.
1994). Further studies are required to determine the
range of genes controlled by Recl6 and to determine
whether they act directly in the meiotic events discussed
here.
A second explanation supposes that Recl6is directly
required for replication, which in turn is required for
recombination and transcription. There are precedents
for the latterevents depending onreplication. Bacteriophage T4 recombination is intimately associated with
replication, and vice uena (MOSIG1994). Mutations that
reduce or delay replication have a recombination-deficient phenotype, much like that of the recl6 mutation.
The double-strand gap-repair model of meiotic recombination supposes that DNA synthesis accompanies recombination (RESNICK 1976; SZOSTAK et al. 1983). In
addition, replication may convert single-strand nicks
into the double-strand breaks apparently required for
meiotic recombination in S. cmevisiae (ROEDER 1995).
UV-induced lesions stimulate mitotic recombination in
a replication-dependent manner in rad1 mutants of S.
cerevisiae (KADYK and HARTWELL
1993). If DNA synthesis
is delayed, recombination may not occur at high levels
because other gene products required for recombination may have been inactivated by the time DNA synthesis occurs. Similarly, transcription may require or be
closely associated with DNA synthesis. Transcription of
genes late in T4 infection requires DNA replication,
apparently because a “sliding clamp” is required to
load onto theDNA-protein complexes for both replication and transcription (HERENDEEN
et al. 1992).Alternatively, replication may make chromatin(in S. pombe)
more accessible to transcription and to recombination.
In this second view Recl6 is primarily required for meiotic replication, and the reel6 recombination- and transcription-deficient phenotypes are a consequence
of the
replication deficiency.
We thank ANDREW
BERGER,
DAWDEVANS,
KAREN HEICHMAN,
PAUI.
NURSE, and GERI PINGUI.for suggestions on flow cytometry; BOB
LEVISand SANDRA
PENNINGTON
for fluorescence microscopy; FRED
PONTICEILI,
YUKANGLIN, PHIIJPPE
S~.LNKASI,
JEFF VIRGIN, and
AMAR
KIAR for strains: BOBBY
BAUM,HOWARD ClARKF., RUBAIDING,DAVII)
EVANS,MARY Fox, YUKANGLIN, and JEFF VIRGINfor unpublished
observations; SUE AMCNIXEN,
JOE FARAH,MARYFox, KAREN HEICHand ANDREW TAYLOR
for comments on the manuMAN, JIM ROBERTS,
script; and KAREN BRIGHTON
and MICHAEI.
MASCHINOT
for skillfully
preparing it. MIKEPARKER
and the HutchinsonBiocomputing Shared
Resource (NCI P30 CA15704) provided image analysis facilities. Y.F.L.
was supported, in part, by funds provided to the Fred Hutchinson
Cancer Research Center by the Sammamish Hills Guild. This work
was supported by Public Health Service grant GM-9‘2194 from the
National Institutes of General Medical Sciences.
Note added in pro$ The S. cereuisiae SPOll gene product
becomes covalently linked to DNA at meitoticallyinduced double-strand breaks (S. KEENEY, C. N. GIROUX
and N. KLECKNER, Cell 88: 375-384, 1997). See also A.
BERGERAT,
B. DE MASSY, D. GADELLE,
P.-C. VAROUTAS,
A.
NICOLASand P. FORTERRE,
Nature 386: 414-416, 1997.
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Communicating editor: M. E. ZOIAN